N-heterocyclic aromatics are environmentally important pollutants, however, little is known of their mechanism of action or biological effects. The objective is to investigate the metabolic activation of 7H- dibenzo(c,g)carbazole (DBC) which is a potent carcinogen in mouse lung, liver, and skin, with respect to dibenz(a,j)acridine (DBA) a carcinogen in mouse lung and skin. Since the structural difference between the well characterized polycyclic aromatic hydrocarbons and the N-heterocyclic analogs is the existence of a nitrogen atom in the ring system of the latter, it is hypothesized that any differences in metabolism, metabolic activation, DNA binding, or carcinogenic potency is due not only to the presence of a nitrogen atom but to the aromaticity of the heteroatom containing ring.
The specific aims i nvolve the characterization of the metabolic activation, DNA binding and biological effects of the lesions produced by DBC and DBA in mouse skin, liver and lung following topical application: 1) Elucidate the mechanism of activation, characterize the major adducts and the binding sites of DBC and DBA to DNA in vitro. 2) Characterize the major adducts of DBC and DBA in lung, liver and skin, both the organic moieties and DNA bases involved, using a combination of in vivo and analytical analyses. 3) Determine the point mutations produced in the ras oncogene for DBC and DBA for specific major adducts in liver and skin. In vitro studies using liver P450 microsomal preparations which catalyze two and one-electron oxidation pathways, and peroxidases which catalyze one-electron oxidation pathways will be used to characterize DNA adducts of DBC and DBA and their proximate metabolites 3- OH-DBC and 3,4-diol DBA. DBC-DNA adduct standards will be synthesized using electrochemical oxidation procedures for DBC in presence of nucleotide bases while DBA-DNA adduct standards will be synthesized by standard procedures using the do epoxide of DBA. A combination of 32P- postlabeling, HPLC, fluorescence, 1H and two dimensional NMR spectroscopy and fast atom bombardment coupled with collisionally activated decomposition mass spectrometry will be used to characterize the major adducts in vitro and in vivo. In vitro studies and syntheses will be used to generate adduct standards for fingerprint of DNA adducts using 32P- postlabel and HPLC for in vivo studies. PCR and DNA sequencing will be used to analyze for point mutations in the target tissues. This approach will provide valuable information concerning the metabolic activation of an important class of carcinogens and their biological consequences and will lead to a better understanding of the mechanism(s) of action of N- heterocyclic aromatic carcinogenesis.
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